Title: Selective Strategies for Mycobacterial Cell Wall Labeling 7. Project Summary/Abstract Up to 2 billion people worldwide are infected by Mycobacterium tuberculosis (Mtb), the bacterial agent of TB. Typical treatments for TB require multiple drugs taken over several months, demonstrating the unique efficacy of Mtb as a pathogen. Studying features essential to the virulence and vitality of Mtb at a molecular level can provide fundamental knowledge about how mycobacteria survive and evolve despite harsh growth conditions and exposure to drug treatment courses. The cell wall of Mtb has been implicated in promoting survival inside lung tissues, and presents a significant barrier to the ability to use diagnostic probes and therapeutic efforts to combat TB. Direct observation of changes in structure and dynamics of the cell wall under conditions relevant to infection (i.e. live macrophage hosts) can provide critical information regarding how this structure responds to drug treatment and the harsh conditions presented by the immune system. This information can be applied towards establishing better methods for diagnosing and treating TB. Bioorthogonal reactions are an attractive technology that enables direct visualization of discrete cellular structures. However, labeling the cell wall of Mtb residing inside host cells is a significant challenge. This proposal seeks to establish imaging strategies using bioorthogonal reactivity for the fluorescent labeling of independent cell wall components of Mtb inside macrophage hosts. Central to this proposal is the ability to use the natural biosynthetic machinery of Mtb to incorporate chemical reporters and fluorophores into the outer glycolipids of the cell envelope. A library of trehalose-conjugated reporters and trehalose-conjugated fluorophores will be designed, synthesized and tested using this metabolic labeling strategy. A similar approach will then be used to fluorescently label peptidoglycan simultaneously with the trehalose glycolipids. Rational molecular design and reaction optimization that enables two simultaneous bioorthogonal reactions to install fluorophores suitable for two- wavelength imaging will be carried out. The invention of a dual labeling strategy will allow for direct imaging of two crucial cell wall components of mycobacteria inside live macrophages. Subsequently, the behavior of these two cell wall components will be investigated as a function of drug treatment and environmental stress using these labeling strategies to directly inform on cell wall structure and metabolism. Through fluorescence microscopy and fluorescence recovery experiments, a model for how multiple cell wall components change over time will be established. Further examination into the defense mechanisms that necessitate multiple drug cocktails to treat TB, as well as how Mtb responds to drug treatments through changes in the cell wall structure, will also be conducted. This research will establish an enabling technology to provide answers to questions regarding the efficacy of Mtb and insight that could lead to improved TB treatment efforts.